The CYC1 and CYC7 genes and their corresponding gene products, iso- 1-cytochrome c and iso-2-cytochrome c in the yeast Saccharomyces cerevisiae are particularly suitable for investigating gene expression, protein synthesis aNd maturation, mitochondrial import and evolutionary relationships. This iso-cytochrome c system will be used to investigate the following processes: a re-examination and determination of the properties of the TATA box elements required for transcription initiation of the CYC1 mRNA; the characterization of the 3' signal for the CYC1 mRNA and the determination if this 3' end is formed by termination or cleavage; the sequences and structures required for translation, including selection of the AUG codon for initiation of translation; factors required for stability of apo-cytochrome c; the amino acid sequences of iso-1-cytochrome c effecting post-translational modification including cleavage of the amino-terminal methionine, acetylation of the amino-terminal residues, and trimethylation of the lysine 77; the residues and regions of iso-1-cytochrome c required for binding to mitochondria and for heme attachment; the role of heme attachment for mitochondrial import; the characterization of the CYC2 gene product, a candidate for the mitochondrial receptor; the identification and characterization of other genes required for binding, heme attachment and the import process; and the pattern of coordinate regulation of CYC1 (iso-1- cytochrome c), CYC7 (iso-2-cytochrome c), CYC3 (cytochrome c heme lyase, required for heme attachment) and CYC 2 (the putative receptor). These studies should reveal the intricate interaction of the many processes required for expression of a eukaryotic gene. In addition, the DNA sequences and characterized genes in the COR and ARC regions, that encompass, respectively, CYC1 and CYC7, will be used to evaluate evolutionary relationships. Also, we are developing a set of mapping strains that can be used to conveniently assign and recessive mutation to its chromosome.